There is a critical need to understand the regulation of cell wall metabolism in Mycobacterium tuberculosis because it contributes to antibiotic tolerance, which exacerbates tuberculosis outcomes. The objective of this proposal is to build a molecular model for how environmental information flows through phosphorylation of three cell wall regulators to dynamically control cell wall metabolism in mycobacteria. The central hypothesis of this proposal is that the central regulators of the cell wall during growth also regulate it in stress. This hypothesis is based on our data that the phosphatase PstP controls growth as well as stress responses, and that the phosphorylated regulators CwlM and DivIVA are required for growth and antibiotic survival. The rationale for this research is that a molecular understanding of cell wall regulation will pave the way for better TB drugs. Aim 1: Determine how the phosphatase PstP orchestrates cell wall metabolism. Our working hypothesis is that phosphorylation of PstP regulates its activity against cell wall factors and helps coordinate the transition from growth to stasis. We will: a) determine how PstP phosphorylation is affected by stresses in Mtb; b) identify the key substrates of PstP and determine the activity of different PstP phospho-isoforms on each substrate; and c) determine how PstP contributes to antibiotic tolerance in Mtb. Aim 2: Determine how CwlM regulates multiple peptidoglycan enzymes. Our working hypothesis is that CwlM is regulated by phosphorylation and recycled peptidoglycan, and in turn regulates peptidoglycan synthesis at multiple steps. We will: a) identify conditions that alter CwlM?s phosphorylation in Mtb; b) characterize the effects of CwlM and CwlM~P on the binding and activity of its interaction partner enzymes; and c) Measure and characterize the function and regulation of the catalytic activity of CwlM. Aim 3: Determine how DivIVA coordinates polar cell wall metabolism. Our working hypothesis is that DivIVA activates cell wall precursor enzymes to promote growth, and is regulated by phosphorylation. We will: a) determine how DivIVA?s phosphorylation is affected by growth and stress conditions in Mtb; b) identify sites on DivIVA required for its protein interactions, and characterize the phospho-dependence of the interactions; and c) measure the effects of DivIVA and DivIV~P on the activity of their interaction partners. Upon completion of this work we expect to have a multi-level molecular model of the signaling pathways that control cell wall precursor synthesis in mycobacteria. We will characterize the regulation of the phosphatase PstP, which is a master regulator and a candidate drug target for both antibiotics and anti-tolerance drugs. We will describe the signaling role of the protein interactions between the intermediate regulators CwlM and DivIVA and their enzymatic regulatory targets; these interactions are potential targets for anti-tolerance drugs. This work will lay the groundwork for novel drug screens.